Spatial distribution and risk assessments due to the microplastics pollution in sediments of Karnaphuli River Estuary, Bangladesh

Microplastics (MPs) have become an emerging global pollutant due to their widespread dispersion and potential threats to marine ecosystems. However, studies on MPs in estuarine and coastal ecosystems of Bangladesh are very limited. Here, we conducted the first study on abundance, distribution, characteristics, and risk assessment of microplastics in the sediment of Karnaphuli River estuary, Bangladesh. Microplastic particles were extracted from sediments of 30 stations along the estuary by density separation and then enumerated and characterized using a stereomicroscope and Fourier Transform Infrared (FT-IR) spectroscopy. In the collected sediment of the Karnaphuli River estuary, the number of MPs varied from 22.29 to 59.5 items kg−1 of dry weight. The mean abundance was higher in the downstream and left banks of the estuary, whereas the predominant shape, colour, and size of MPs were films (35%), and white (19%), and 1–5 mm (30.38%), respectively. Major polymer types were polyethylene terephthalate, polystyrene, polyethylene, cellulose, and nylon. MPs were found to pose risks (low to high) in the sediment of the estuary, with the highest risk occurring at one station near a sewage outlet, according to the results of risk analyses using the pollution risk index, polymer risk index (H), contamination factors, and pollution load index (PLI). The single value index, PLI, clearly demonstrated that all sampling sites were considerably polluted with microplastics (PLI > 1). H values showed toxic polymers, even in lower proportions, possess higher polymeric hazard scores and vice versa. This investigation uncovered new insights on the status of MPs in the sediments of the Karnaphuli River estuary, laying the groundwork for future research and control of microplastic pollution and management.


Materials and methods
Study sites. Karnaphuli is the second-largest estuary in Bangladesh. The Karnaphuli river flows from the Lushai hills in Mizoram, India, and has a watershed area of nearly 11,000 km 2 2,31 ; and travels 180 km of Rangamati mountainous wilderness, Bangladesh. Further, the Karnaphuli River estuary flows about 170 km via the port city of Chittagong and ends in the Bay of Bengal 32 . The estuary is abundant with semidiurnal tides of 2-4 m range with a mean depth of 8-10 m in the external zone of the river 33 . The environmental behavior of the Karnaphuli River estuary changes periodically due to the Indian monsoon 31 . Because of the seasonal wind movement, the climatic conditions of the riverine area in Chittagong vary from season to season. The monsoon season is hot, gloomy, and oppressive, whereas the dry season is warm, generally clear, and humid. The temperature ranges from 58 °F to 90 °F, with an annual rainfall of 17.8 inches 32 . The entire river watershed is geologically composed of tertiary rocks covered with alluvial deposits, with successive layers of mud and sand 34 . Sample collection and processing. Sediment samples were collected across upstream, midstream, and downstream of the river. From July 2020 to May 2021, 90 sediment samples were collected using an Ekman dredge at low tide from 30 locations on the left and right banks of the Karnaphuli River estuary in Bangladesh (Fig. 1). All sediment samples were collected in replicates. Sediment samples, nearly 1 kg weight, were processed at the Bangladesh Oceanographic Research Institute laboratory. Each sediment sample was frozen and dried before sieving with 5 mm to remove larger plastic particles 35 36 . Sediment samples were first homogenized with a stainless-steel spoon before being dried at 90 °C for 24 h. Dried samples were mixed with 300 mL of ZnCl 2 (1.5 g /mL) salt solution 37 and stirred continuously. After that, all floating solids were sieved using 0.3 mm sieve and kept in 20 mL each of 30% H 2 O 2 and FeSO 4 (0.05 M) solution for oxidation of organic materials [38][39][40] . The wet peroxide oxidation treatment was performed to remove organic contaminants at approximately 75 °C for 5 min and boiled till no organic material was left. Then, 6 g sodium chloride (NaCl) per 20 mL of sample solution using density separation method and heated at 75 °C to dissolve sodium chloride entirely and placed to settle using density separator for 24 h. Floating solids were collected using a 0.3 mm custom sieve, and the density separator was rinsed thoroughly with distilled water to transfer all. The solution from the density separator was filtered using 0.45 μm mesh size cellulose nitrite filter paper 41 .
Contamination factors, pollution load index, and polymeric and pollution risk assessments. MPs contamination factor (CF), pollution load index (PLI), pollution risk index (PRI), and polymeric risk assessment (H) in the river sediment were estimated as described in previous studies 24,43,44 . Tomlinson et al. 45 proposed the contamination factors (CF) and pollution load index (PLI) to assess the pollution level in natural ecosystems. CF, PLI, PRI, and H were categorized as values from low contamination to very high contamination, described in Table 1 24,43,44 .  www.nature.com/scientificreports/ The PLI is regarded as a standardized protocol for assessment and monitoring the extent of pollution between different areas 45 . Sampling sites are considered to be polluted, as PLI > 1 45 . The risk assessment model was as follows 45 : where i denotes a station, n presents the number of stations, C i represents MPs abundance at ith station, and C o is the minimum baseline concentration in rivers. Though, the least MPs abundance found in this investigation was considered as minimum baseline concentration due to the lack of previous background data in the same environments and the analytical context of this study.
Xu et al. 46 evaluated that MPs concentration and chemical composition need to be considered to evaluate the potential risks in surface sediments 46 . Lithner et al. 47 assigned hazard scores to compute polymeric risks based on their toxicity level in respective ecosystems. Polymeric risks can be estimated using chemical toxicity coefficients or risk scores (S j ) for the identified polymers in the sediment samples. The hazard scores for MP polymers identified were PS = 30; PE = 11; PET = 4; and Nylon = 50, while cellulose was not available and was excluded in the calculation. Equations (3) and (4) presented polymeric risk assessment for different sampling sites and the entire Karnaphuli River Estuary, Bangladesh, respectively. P ji denotes the number of and S j presents risk score for each single MPs polymer recognized at ith station 24,47 , whereas H river is calculated as the nth root of the polymer risk indices products (Eq. 4), as follows: To compute MPs pollution risk index (PRI), Kabir et al. 44 developed a pollution risk formula for particular stations as well as an entire river; Eqs. (5) and (6) are described as follows: Quality control. All equipment needs to be rinsed using de-ionized water before and after use. All stock solutions were filtered using 0.45 μm mesh size filter paper before use to avoid MPs contamination. Also, all the glassware was rinsed thrice with purified water. All samples were kept covered with aluminum foil or glassware whenever possible or under analysis to avoid external MPs contamination. Three blank samples without salt were analyzed simultaneously to correct any possible MPs contamination from sample processing. No MPs were found in blank samples. Polyester-type clothing was avoided to ensure intentional contamination of microplastics in the samples, and during handling, cotton-made laboratory aprons and nitrile gloves were used. All non/ plastic sieves were washed and sonicated properly, before and after use. All MPs samples were kept in Petri dishes and appropriately covered with aluminum foil; then, all Petri dishes were placed in a glass desiccator to avoid airborne MPs contamination.
Statistical analysis and image preparation. Data were expressed in mean values with their respective standard deviation for better interpretation of MPs pollution. Statistical analyses were performed using R (Studio v.1.1.453, PAST), whereas graphics elaboration was conducted on Prism 8.0 (Graph Pad Software Inc., USA), and ArcGIS 10.6 was used for MPs mapping. One-way analysis of variance (ANOVA) was performed, using Origin Pro, between two or more samples of both left and right banks for investigating their significance (significance level of 0.001). Principal component analysis (PCA) was also performed between independent indicators, in which raw data for MPs abundance on both right and left banks were analyzed to establish a relationship between MPs and sampling sites. Besides, a systematic cluster, i.e., the Heat map analysis method was performed for sampling sites to determine pollution characteristics and sources of pollution.
(1)  (Fig. 2a). However, MPs abundance increased with the downstream and observed maximum at the S15 sampling site. All three sites were in the mouth of the river, which is connected to the sea directly. These results showed that MPs abundance in the sediment increased towards the mouth of the river estuary. This condition might emerge due to the estuary position, which is influenced because of fluctuating currents, tides, and direct access to the sea 48,49 . MPs particle distribution path and trajectory are influenced due to hydrodynamic force, i.e., flow velocity in the estuary as it enters the marine environment 50 . Therefore, the estuaries are prone to MPs contamination and should be considered hotspots for MPs sinks 51,52 .
The main reasons behind significant MPs pollution in Karnaphuli river estuary regions are high industrial effluents and municipal sewage generation rate 53,54 . However, there are no MPs pollution studies in the neighboring areas. Insufficient and inadequate infrastructure and sewage and industrial waste management services have caused a high abundance and disposition of MPs. Still, lack of community involvement has become an additional significant concern in reducing solid waste. For example, Chowdhury et al. 54 reported that municipal solid waste increased from 538 tons/day in 1999 to 1890 tons/day in 2009 due to inadequate segregation of municipal solid waste in residential communities of Chittagong City. Also, the World Bank Group 55 reported that municipal solid waste management service in Bangladesh is yet to be improved with suitable infrastructure and services. Therefore, Mediana and Gamse 56 observed that significant quantities of solid waste, including plastic waste, be disposed of into rivers of Bangladesh. Furthermore, Karnaphuli River Estuary receives solid waste disposal sites from densely populated human settlements and industrial activities in Chittagong City 54 .
Existing riverine MPs research investigated spatial mapping of plastics across river channels. Microplastics enter the Karnaphuli River estuary from various point and non-point (or diffuse) sources. Point sources are from direct discharge, e.g., wastewater effluents, drainage ditches, agricultural runoff, and storm drains, whereas non-point sources are littering, spread over large areas 57 . The high abundance of MPs at the river downstream is significantly influenced by massive discharge by wastewater treatments and sewage effluents via agricultural, industrial, and urbanized areas due to population density and catchment size (Fig. 3). Moderate MPs pollution was observed frequently in sediments, from sampling sites of S1, S2, S4, and S5 due to household activities, microbeads, commercial fishing, etc. Assessing MPs from wastewater treatment plants, the equivalent population serviced and utilized via treatment methods, e.g., tricking filters, activated sludge, etc., needs to be considered 58 . Besides, huge amounts of MPs are consistently observed in close proximity to plastic manufacturing and industrial units. Andrady 8 reported that MPs observed in terrestrial and aquatic ecosystems mainly belong from blast/raw materials used in industrial processes, either from unregistered discharge or accidental leakages. Several studies investigated the abundance of MPs in riverine sediments 59,60 . MPs variations between banks of river estuary were analyzed using one-way Analysis of Variance (ANOVA). Which was paired using Tukey's HSD test. Results showed that MPs abundance were not significantly differed (F = 0.2465, df = 1, p < 0.01) among fifteen sampling sites (Table 2). Tukey's test indicated that the difference of the means is not significant at the significance level of 0.01.
Storm drains are also pointing sources for plastic debris, where MPs originate from road markings and car tires abrasion in urban areas 61,62 . Therefore, Horton et al. 57 reported MPs originated from vehicles in River   Table 3 and observed MPs abundance from few to thousands of particles per kilogram.
Polymer types. Polymeric characteristics of MPs were observed using FTIR analysis, which identified various polymers, such as cellulose, polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), and nylon (Fig. 8). Based on FTIR analysis, PS, PE, PET, cellulose, and nylon accounted for 19.59, 16.66, 27.78, 17.05, and 17.92%, respectively (Fig. 9). PET was the maximum abundant polymer compared to PS, PE, cellulose, and   Color. MPs appearance makes unintentional ingestion by biota due to their possibly ingested, due to their resemblance with original prey or foodstuffs 52,83 . In Karnaphuli River Estuary, MPs of various colors are observed, like red, white, black, green, brown, yellow, orange, pink, gray, blue, and transparent (Fig. 10). Few studies have reported aquatic organisms to resemble foodstuffs with attractive MPs and make them available for ingestion due to their small particle sizes and high buoyancy in aquatic ecosystems 68,84 . White MPs were found maximum (19.25%) in the river estuary, followed by red, black, blue, transparent, green, etc., as shown in Fig. 10. It is observed that MPs with various colors sourced from plastics used in daily life, for example, clothes, packaging, fishing nets, etc., settled primarily on sediments 85,86 . On the other hand, MPs color also alters due to weathering during transport in aquatic ecosystems, surface water, and marine materials 87,88 . Therefore, colored MPs in the Karnaphuli River estuary recommend that these MPs originate from synthetic and organic materials; after that, in-depth investigations for detailed sources are required in the Karnaphuli River estuary.

Source identifications.
Because of the proportions of various plastic types at different sample sites, using principal component analysis (PCA) to analyze the prevalence of MPs can be more understandable. PCAs of eight different MPs describe their distributions in the sediment of Karnaphuli River Estuary shown in Fig. 11a, which revealed the substantial differences via their distributions from each sampling site 89 . Results revealed that different MPs shapes dominate different sampling sites in the Karnaphuli River estuary. The first principal component analysis (PCA1) has shown robust contributions (48.3%) for fragments, films, pellet, and foam observed widely in sediments of many sampling sites in the Karnaphuli River estuary. Whereas second component (PCA2) showed granules, fiber, line, and flakes are dominated (17.2%) in sediments but possesses less distribution compared to PCA1, in which most MPs are originated from activities, like household, fishing, and agricultural practices, along with chemical and clothes industries. Thus, distribution characteristics have shown agricultural activities as the primary source for films in the river estuary. The eight parameters (shapes) were distinctly categorized into two groups, and heat map analysis was used to distinguish sampling sites with identical shape distributions (Fig. 11b). Results from the Karnaphuli River estuary indicated that foam, films, fragments,   , S10 (40.40), S12 (37.72), S13 (40.39), S14 (52.57) and S15 (63.29) have shown PRI < 150. Besides, in downstream sampling sites, S12-S15 spread with industries, agricultural, residential, and urban land uses. For the entire study area, the PRI was 43.55, which indicates river estuary overall under the low-risk category. Therefore, in this study, as polymers are source-specific, as a result, land-use behavior affects the sources of polymers and results in high MPs pollution posed by point and non-point sources of MPs from various land-uses based on polymeric types as well as their toxicity. Notably, polymeric risk assessments provided insights into the richness of toxic polymers in river environments. MPs polymer risk index (H) for all sampling sites are presented in Fig. 12b. The results showed that polymeric risks varied low to high from upstream to downstream. Based on the risks classification, only samples from S11 site showed considerable risks with polymeric risk assessment of 347, which indicated high-risk category (Class III) and revealed the presence of toxic polymers that results in higher hazard scores (Nylon 6: 50; PS: 30), along with the stations 47 . Even though highly toxic polymers were observed but in lower proportions, compared to polymers that possess higher polymeric hazard scores. Higher proportions of polymers with relatively low hazard scores resulted in lower polymeric risk values, for example, PS: 11; PET:4. However, polymeric risk assessment (H) for sampling site S2 showed low polymeric risks (14.06) of Class II from the entire river estuary.
MPs CF results clearly showed that all sampling sites in the river were significantly polluted with MPs (1 ≤ CF < 3) and indicated similar pollution levels at all sampling stations and are moderately contaminated with high MPs abundance (Fig. 12c). The overall pollution load index is 1.73 for the river estuary, which shows PLI > 1, indicating MPs pollution for all river sediment samples analyzed in the Karnaphuli River estuary. However, by stations, CF followed the decreasing order of contamination as S15 > S14 > S13 > S10 > S8 > S12 > S9 > S11 > S6 > S3 > S7 > S5 > S1 > S4 > S2. Contamination of sediment by microplastics is undesirable as it can accumulate in the fish and other organisms, causing deleterious effects.

Conclusions
Estuaries are crucial ecosystems to understand the fate and transport of MPs to the ocean via land-based sources. This was the first study on MPs pollution from Karnaphully River estuarine ecosystem in Bangladesh. In this study, a high abundance of MPs was observed in the Karnaphuli estuary compared to other most of the estuaries in the Asian region and found gradually higher levels towards the mouth of the estuary. Film-shaped, white-colored, and larger-sized (1000-5000 μm) MPs were dominant in the Karnaphully River estuary. Among the polymer types, polyethylene terephthalate was most abundant. The pollution load and risk indices showed that all sampling sites across river banks were polluted with MPs and posed significant risks to the ecosystems. Land-use behavior such as agricultural runoff, industrial effluents, household & fishing activities, and urbanized locality dominate polymeric abundance. This study provides a database baseline for microplastic pollution in www.nature.com/scientificreports/ the Karnaphully River estuary due to anthropogenic activities. Thus, policymakers, scientists, ecologists, social environmentalists, and hydrologists are based on the database non-government organizations, government, etc., can strategically plan for river estuary conservation and management.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.